肠出血性大肠杆菌O157∶H7变种Q蛋白对志贺毒素表达的影响

Effect of Protein Q on Shiga Toxin Expression in Enterohemorrhage Escherichia coli O157 Variant

1株肠出血性大肠杆菌O15 7∶H7变种EC169菌株,携带stx基因但不表达志贺毒素。通过高效热不对称交错聚合酶链式反应(high-efficiency thermal asymmetric interlaced polymerase chain reaction,hiTAIL-PCR)hiTAILPCR扩增得到EC169 stx1及其上游核苷酸片段并克隆测序,结果表明:EC169 q基因与标准株sakai q基因相比存在6个SNP位点。通过PCR扩增O157∶H7高毒株EC150 q基因全长,并构建表达载体pkk223-q分别转化EC169和低毒株EC157。反转录荧光定量PCR实验结果表明,外源q基因在EC169和EC157重组菌中可高效表达,并引起EC157stx转录水平上调,但EC169重组菌stx转录水平不变。反向乳胶凝集实验结果亦证实EC157重组菌志贺毒素表达量提高,而EC169重组菌志贺毒素表达量不变。Q蛋白变异可能并非EC169志贺毒素不表达的主要原因。

An enterohemorrhagic Escherichia coli(EHEC) O157: H7 strain EC169 carrying both stx1 and stx2 genes but not producing Shiga toxins was used in this study to investigate the reason for these characteristics. The stx1 gene and its upstream region from EC169 were amplified by hiTAIL-PCR. Sequencing data showed that EC169 q gene had 6 single nucleotide polymorphism(SNP) sites compared with the corresponding region of the typical strain sakai. A highly virulent O157:H7 strain EC150 and a hypovirulent O157: H7 strain EC157 were tested in this study. The full length of EC150 q gene was amplified and connected to the expression vector pkk223. The recombinant plasmid was then transformed into EC169 and EC157, respectively. The qRT-PCR results showed that the pkk223-q vector was efficiently expressed in the two recombinant strains, respectively. qRT-PCR and RPLA results demonstrated that the level of Shiga-toxin expression was improved in EC157 recombinant strain, but its expression was not changed in EC169 recombinant strain. These results suggest that the SNP variation of protein Q might not be responsible for the non-expression of Shiga-toxins in EC169, suggesting that other mechanisms may be involved in controlling the expression of Shiga-toxins in EC169. This study might contribute to study the control and regulation of Stx phage.